In the beginning of 1980s, the group of Sporn, Roberts, et al. isolated and purified TGF-α from a culture supernatant of murine sarcoma virus-transformed mouse 3T3 cells as one of two factors necessary to induce colony formation of normal rat kidney fibroblast cells (NRK cells) in soft agar (NPLs 1 to 6).
TGF-α is biosynthesized as a precursor polypeptide consisting of 160 amino acids. After glycosylated and palmitoylated inside a cell, TGF-α is expressed on the surface of the cell membrane as a type I transmembrane protein (transmembrane TGF-α) (NPL 7). Then, the extracellular domain is cleaved by a metalloprotease such as TACE/ADAM17. Hence, a single polypeptide consisting of 50 amino acids from 40th to 89th amino acids (approximately 6 kDa) is released as secreted TGF-α (NPLs 8 to 10). Secreted TGF-α has a characteristic structure in which disulfide bonds are formed at three sites by six cysteine residues (NPLs 11 to 13). This structure was first discovered in epidermal growth factor (EGF), and thereafter is called an EGF-like domain. There are 13 types of proteins having an EGF-like domain in total, including, other than TGF-α and EGF, amphiregulin, HB-EGF, β-cellulin, epiregulin, epigen, neuregulin-1, neuregulin-2, neuregulin-3, neuregulin-4, neuregulin-5, and neuregulin-6 (hereinafter, neuregulin-1 to -6 are referred to as neuregulins). These are collectively called EGF family molecules (NPLs 10, 14). All of the EGF family molecules are biosynthesized as the type I membrane proteins like TGF-α. Releasable extracellular domains are formed by shedding with a protease (NPL 10). There is a high degree of amino acid homology among species, and the EGF-like domain structure is also preserved. For example, human TGF-α has an amino acid homology of 92% with mouse TGF-α, and human TGF-α works in mice and rats (NPL 15).
EGF family molecules act as ligands that directly bind to the receptor tyrosine kinase EGF receptor (EGFR) family (also known as ErbB family). Currently, four types are identified in the EGFR family based on the structural similarity, and respectively called EGFR (ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4) (NPLs 14, 16). Among 13 types of existing EGF family molecules, TGF-α, EGF, amphiregulin, β-cellulin, epiregulin, and the like are known as ligands which bind to EGFR. Neuregulins bind to HER3, while neuregulins, β-cellulin, and HB-EGF bind to HER4. HER2 has no ligand-binding site (NPLs 14, 16).
When an EGF family molecule binds to a receptor EGFR family molecule, the receptor forms a dimer, and phosphorylation of tyrosine residues is induced by tyrosine kinases located in the intracellular domain (NPL 16). Subsequent to this, various proteins in the cell are activated like a cascade. As typical examples of such a cascade pathway, the Ras/Raf/MAPK pathway, the PI3K/Akt/mTOR pathway, and the JAK-STAT pathway are known. The Ras/Raf/MAPK pathway is mainly associated with cell proliferation and survival, PI3K/Akt/mTOR pathway is associated with cell growth, anti-apoptosis, cell infiltration, cell migration, and so forth (NPL 17).
When secreted TGF-α is added to normal rat fibroblast cells Rat-1 in soft agar, the cell form of Rat-1 is changed to a transformed cell-like form, and colonies are formed in the agar (NPL 15). Moreover, CHO cells having a TGF-α gene introduced thereinto also become transformant-like cells, and readily form a tumor when transplanted into a nude mouse (NPL 9). In this event, EGFR on the surface of the CHO cells is significantly phosphorylated. Accordingly, it is understood that TGF-α secreted by CHO induces cell proliferation in an autocrine manner. As another physiological function of TGF-α, an action as a potent angiogenesis-inducing factor is known. It is believed that TGF-α acting on EGFR expressed in vascular endothelial cells induces migration and proliferation of the vascular endothelial cells (NPLs 18 to 21).
The distribution of TGF-α in the body of a healthy subject is characterized by the expression in mucosal epithelial cells such as in the respiratory epithelium and mucosal epithelium of the large intestine (NPLs 22 to 24). This expression pattern resembles that of EGFR. Meanwhile, as to cancers, it is well known that TGF-α is over-expressed mainly in solid cancers (NPLs 22, 23). Enhanced expression of TGF-α at mRNA and protein levels has been observed in various carcinomas such as head and neck cancers, lung cancer, breast cancer, stomach cancer, colorectal cancer, kidney cancer, liver cancer, ovarian cancer, and melanoma. Overexpression of the receptor EGFR has also been observed in various cancer tissues. It has been reported amounts of TGF-α and EGFR expressed are positively correlated particularly in non-small cell lung cancer, head and neck squamous cell carcinomas, and kidney cancer (NPLs 25 to 29). Developed at a site nearby a solid cancer is a tissue called an interstitial tissue which is constituted of a stroma (mainly fibroblast cells), serving as the scaffold for cancer cells, and a blood vessel for transporting nutrients. The result of immunohistostaining of cancer tissues using an anti-TGF-α antibody has revealed that TGF-α exists not only in a cancer site but also in an interstitial tissue (NPLs 25, 29). From these, it is believed that TGF-α not only induces the proliferation of cancer cells in an autocrine manner, but also contributes to the malignant transformation of the cancer by functioning as a growth factor of the interstitial tissue supporting the cancer growth (NPLs 18 to 21, 30).
As described above, there is no doubt that EGF family molecules including TGF-α and the receptor EGFR are involved in proliferation of cancers. Accordingly, as a matter of course, it is expected that cancer cell proliferation can be controlled by targeting TGF-α and EGFR (NPLs 16, 17, 31).
Research for a monoclonal antibody targeting EGFR in cancer treatment has been started since the beginning of 1980s (NPLs 32 to 34). Cetuximab (trade name: Erbitux) was developed as a monoclonal antibody which has an activity of inhibiting tyrosine phosphorylation and dimerization of EGFR by binding thereto competitively with the ligand EGF (NPLs 35, 36). Cetuximab was confirmed to suppress proliferation of cultured cancer cells such as A431 cells, and to demonstrate an anti-tumor effect in a cancer-bearing mouse model, also. In 1990, clinical trial had been started targeting patients with squamous cell lung carcinoma. In a randomized controlled trial conducted in 2001 and 2002, the effect on colorectal cancer was proved. In 2003, cetuximab was approved for the first time in Switzerland as a therapeutic agent against metastatic colorectal cancer. FDA also approved cetuximab in 2004 as a therapeutic agent against metastatic colorectal cancer expressing EGFR, and additionally approved in 2006 as a therapeutic agent against head and neck cancers expressing EGFR. In Japan, the Ministry of Health, Labour and Welfare approved the manufacturing and sales of cetuximab in 2008 as a therapeutic agent against EGFR-positive, advanced and recurrent colon and rectal cancers uncurable by resection (NPL 37). Panitumumab (trade name: Vectibix, Amgen Inc.) is known as an anti-EGFR monoclonal antibody which suppresses cancer proliferation in a similar manner by binding to EGFR(NPL 38). In Europe and the United States, panitumumab is used as a therapeutic agent against EGFR-positive, advanced and recurrent colorectal cancers similarly to cetuximab. Both of the anti-EGFR blocking antibodies improve the overall survival time and progression-free survival time of colorectal cancer patients uncurable by chemotherapy, and may preserve the quality of life. Thus, it is desired to broaden the application to other EGFR-positive cancers (NPLs 31, 35, 39).
However, on the other hand, colorectal cancer becomes cetuximab resistance at a high frequency, and progress of colorectal cancer has been observed in 50% or more of patients (NPLs 40, 41). It is pointed out that K-Ras gene mutation is involved in this cetuximab resistance. In other words, if there is a mutation in the gene of K-Ras located downstream of EGFR, cells are activated and the cancer keeps proliferating regardless of whether cetuximab or panitumumab blocks EGFR or not (NPL 42).
In 1960s, K-Ras and H-Ras were isolated as oncogenes from Kirusten rat and Harbey sarcoma viruses, respectively. Then, activated H-Ras was isolated from a human bladder cancer cell line, activated K-Ras was isolated from a human lung cancer cell line, and activated N-Ras was isolated from a neuroblastoma (NPLs 43, 44). The homology of gene products from these three types of Ras gene is as high as approximately 85%. A Ras gene product in normal cells is one kind of low-molecular-weight G protein having a GTPase activity, and plays a role in progress of cell proliferation. If a point mutation occurs in the Ras gene, a missense mutation may occur in an amino acid to be encoded, lowering the original GTPase activity in some cases. An association is suggested between tumorigenesis and a point mutation, particularly, at the glycine residue of codon 12 or 13. Mutations at codon 12 or 13 have been observed in approximately 40% of colorectal cancer patients (NPL 40). It is believed that in order to maintain the active state of the mutated Ras protein to which GTP binds, signals are constantly transferred downstream, causing abnormal cell proliferation and tumorigenesis (NPL 45). In this event, various EGF family molecules including TGF-α are over-expressed. This presumably gives an impetus to cancer cell proliferation, and enhances cancer enlargement (NPL 46).
When there is a K-Ras gene mutation, no effect is expected even from the use of cetuximab or panitumumab, and only a risk of the side effect remains. For this reason, the use of these therapeutic drugs is restricted in Europe under such a condition that the drugs are used only for a patient having no K-Ras gene mutation. In Japan also, it is recommended that when cetuximab is used, the target is a patient having normal K-Ras gene who is expected to respond. As other EGFR inhibitors, gefitinib (trade name: Iressa) and erlotinib (trade name: Tarceva) are known, which are low molecular weight drug for inhibiting the action of tyrosine kinases present in the intracellular domain. However, there has been reported that the outcome of these therapeutic drugs is also unsatisfactory to a patient having a K-Ras mutation (NPLs 39, 47, 48).
Meanwhile, as long as we know, there is no report that a monoclonal antibody targeting TGF-α located upstream of EGFR inhibits proliferation of cancer cells derived from an actual cancer patient or controls tumor formation in a cancer-bearing model mouse into which human cancer cells are transplanted.
Nevertheless, there is a case in which a monoclonal antibody against TGF-α was used to control colony formation of cultured cells seeded in soft agar. In 1986, Rosenthal et al. conducted the following experiment using a monoclonal antibody TGF-α1 against human TGF-α. Although normal rat fibroblast cells Rat-1 hardly form colonies in soft agar, colony formation is readily induced by addition of human TGF-α. Moreover, clone 16 and clone 42 of a Rat-1 cell line established by forcibly introducing a human TGF-α gene thereinto also acquire colony-forming ability. The anti-TGF-α antibody TGF-α1 added to this culture system inhibited the colony formation. On the other hand, Rat-1 into which an activated Ras gene was introduced acquired colony-forming ability. However, TGF-α1 of the anti-TGF-α antibody added to this culture system was not able to inhibit the colony formation (NPL 15).
A similar experiment was conducted by Ciardiello et al. in 1990 (NPL 49). A TGF-α gene was introduced into normal mammary epithelial cells MCF10A to establish TGF-α-overexpressing cell lines MCF10A TGF-α C13 (hereinafter referred to as C13) and MCF10A TGF-α C14 (hereinafter referred to as C14). MCF10A cells of the parental line hardly form colonies in soft agar, while C13 and C14 have a high colony-forming ability. An anti-TGF-α monoclonal antibody Tab1 added to the culture system suppressed colony formation of C13 and C14 in an antibody concentration-dependent manner (the colony formation was inhibited by approximately 90% at an antibody concentration of 50 μg/mL or higher). In this experimental system, EGFR blocking antibody clone 528 achieved a colony formation inhibition 10 times or more as strong as Tab1 when evaluated by antibody concentration (the colony formation was inhibited by 90% or more at an antibody concentration of 5 μg/mL or higher).
Cell lines MCF10A Ha-ras C11 and MCF10A Ha-ras C12 established by introducing an activated Ras gene into MCF10A overexpress TGF-α and acquire a high colony-forming ability. These activated Ras-introduced cells show a strong resistance to the EGFR blocking antibody and the anti-TGF-α antibody in comparison with the above-described TGF-α introduced cells. EGFR blocking antibody 528 achieved only approximately 60 to 70% inhibition of the colony formation at an antibody concentration of 5 μg/mL. Meanwhile, the anti-TGF-α antibody Tab1 only achieved approximately 50% inhibition of the colony formation even at an antibody concentration of 50 μg/mL to 100 μg/mL.
In 1989, Sorvillo et al. of Oncogene Science, Inc. received a patent for multiple anti-TGF-α monoclonal antibodies (PLT 1). The patent specification describes the reaction specificity to TGF-α of clone 213-4.4, clone 134A-2B3, and clones 137 to 178, but does not provide data on these antibodies inhibiting TGF-α physiological function or inhibiting proliferation of TGF-α-expressing cells. In 1990, the same research group reported, in an article, anti-TGF-α antibodies described in the patent specification (NPL 50). This article illustrates that clones 189 to 2130 have an effect of inhibiting TGF-α binding to EGFR, but does not mention the influence, or the like, on proliferation of TGF-α-expressing cells and Ras mutated cancers.
As described above, as to controlling cancer cell proliferation, it has not been expected so far that an antibody against TGF-α present upstream of EGFR demonstrates a superior effect to an EGFR inhibitor including an EGFR blocking antibody. Further, it has been regarded as common sense so far that in cancer cells which are constantly activated on a downstream side of EGFR including Ras, it is still difficult to suppress the cancer cell proliferation even when EGFR located upstream and TGF-α located further upstream are blocked (NPLs 31, 46).
It has been reported that Ras gene mutations occur in quite a wide range of tumors. Among all the human cancers, 17 to 25% of patients on average have K-Ras gene mutations (NPL 51). Particularly, gene mutations were found in 35 to 40% of colorectal cancer patients, 35% of lung cancer patients, 55% of thyroid cancer patients, and 80 to 90% of pancreatic cancer patients (NPLs 52, 53). As described above, an activated Ras gene mutation causes a strong resistance shown to various EGFR inhibitors including cetuximab and panitumumab (NPLs 42, 54). A development of a therapeutic drug against Ras mutated cancers highly resistant to available drugs is an importantissue directly involved in improvement of living of human kind, and is also strongly expected by many cancer patients and doctors. However, there has been no effective means available for treatment of Ras mutated cancers up to now.